title: Methods for analyzing peptides and proteins on a chromatographic timescale by electron-transfer dissociation mass spectrometry.
abstract: Advancement in proteomics research relies on the development of new, innovative tools for identifying and characterizing proteins. Here, we describe a protocol for analyzing peptides and proteins on a chromatographic timescale by coupling nanoflow reverse-phase (RP) liquid chromatography (LC) to electron-transfer dissociation (ETD) mass spectrometry. For this protocol, proteins can be proteolytically digested before ETD analysis, although digestion is not necessary for all applications. Proteins <or=30 kDa can be analyzed intact, particularly if the objective is protein identification. Peptides or proteins are loaded onto a RP column and are gradient-eluted into an ETD-enabled mass spectrometer. ETD tandem mass spectrometry (MS/MS) provides extensive sequence information required for the unambiguous identification of peptides and proteins and for characterization of posttranslational modifications. ETD is a powerful MS/MS technique and does not compromise the sensitivity and speed necessary for online chromatographic separations. The described procedure for sample preparation, column packing, sample loading and ETD analysis can be implemented in 5-15 h.
authors.affiliation: University of Virginia, Department of Chemistry, McCormick Road, Charlottesville, Virginia 22904, USA.
authors.forename: Namrata D
authors.initials: ND
authors.lastname: Udeshi
authors.forename: Philip D
authors.initials: PD
authors.lastname: Compton
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
authors.forename: Kristie L
authors.initials: KL
authors.lastname: Rose
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D007477:Ions / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000737:chemistry
chemical_list: D007477:Ions; D010455:Peptides; D011506:Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; C514860:rev protein, Human Immunodeficiency Virus-1